Terminally modified RNA

Abstract

The invention relates to compositions and methods for the manufacture and optimization of modified mRNA molecules via optimization of their terminal architecture.

The invention claimed is: 
     
       1. A mRNA comprising
 (a) a 5′ untranslated region (5′UTR); 
 (b) a region of linked nucleosides encoding a polypeptide of interest; 
 (c) a 3′ untranslated region (3′ UTR) comprising at least one microRNA binding site; and 
 (d) a 3′ tailing region of linked nucleosides, 
 wherein each uridine in the mRNA is a modified uridine nucleoside. 
 
     
     
       2. The mRNA of  claim 1 , wherein the modified uridine nucleoside is a pseudouridine analog. 
     
     
       3. The mRNA of  claim 2 , wherein the pseudouridine analog is a 1-methyl pseudouridine. 
     
     
       4. The mRNA of  claim 3 , wherein each cytidine in the mRNA is 5-methyl cytidine. 
     
     
       5. The mRNA of  claim 1 , wherein the 5′UTR comprises a translation initiation sequence selected from the group consisting of Kozak sequence and an internal ribosome entry site (IRES). 
     
     
       6. The synthetic isolated terminally optimized mRNA of  claim 1 , comprising at least one 5′ cap structure. 
     
     
       7. The mRNA of  claim 6 , wherein the at least one 5′ cap structure is selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, and CAP-003-CAP-225. 
     
     
       8. The mRNA of  claim 1 , wherein the at least one microRNA binding site is for an immune cell specific microRNA. 
     
     
       9. The mRNA of  claim 8 , wherein the immune cell specific microRNA is selected from the group consisting of miR-142-3p, miR-142-5p, miR-146a and miR-146b. 
     
     
       10. The mRNA of  claim 1 , wherein the 3′ tailing region of linked nucleosides further comprises a chain terminating nucleoside. 
     
     
       11. The mRNA of  claim 10 , wherein the chain terminating nucleoside is selected from the group consisting of 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine, 2′,3′-dideoxynucleosides, 2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, 2′,3′-dideoxythymine, a 2′-deoxynucleoside, and —O— methylnucleoside. 
     
     
       12. The mRNA of  claim 1 , wherein the 3′ tailing region comprises a stem loop sequence. 
     
     
       13. The mRNA of  claim 1 , wherein the modified uridine nucleoside is 1-methyl pseudouridine, and wherein each cytidine in the mRNA is 5-methyl cytidine. 
     
     
       14. The mRNA of  claim 1 , wherein the 3′ tailing region of linked nucleosides comprises a poly A tail of at least 100, at least 120 or at least 140 nucleosides. 
     
     
       15. The mRNA of  claim 1 , wherein the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody or fusion protein.